Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.

Más P, Beachy RN - J. Cell Biol. (1999)

Bottom Line:
At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER.Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection.MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

ABSTRACTLittle is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

Figure 11: Model of TMV infection in BY2 protoplasts. (1) Early in infection, TMV vRNA comes in close association with membranes of the ER, and vRNA-replicase complexes associated with ER are transported via microtubules to perinuclear positions. (2) Nascent vRNAs synthesize MP that remains associated with vRNA. (3) Formation and anchoring of large ER-derived structures containing MP, replicase, and vRNA that are stabilized by MP and microfilament interactions. (4) Microtubule-based transport system of vRNA-MP complexes towards the periphery of the cell to initiate cell-to-cell spread. (5) Protrusion of ER containing vRNA and MP through the plasma membrane. Note that the model is not drawn in scale.

Mentions:
The data presented here and in previous publications are consistent with a model of TMV infection (Fig. 11) in which the replication of vRNA takes place in close association with membranes of the ER (Fig. 11, Fig. 1). In this model, cytoskeletal elements are involved in targeting vRNA/replicase complexes to the perinuclear ER, perhaps via a retrograde flow of microtubules with ER attached as cargo. The ER-associated nascent vRNAs in replication complexes function as mRNAs for the synthesis of MP (Fig. 11, Fig. 2). The MP remains associated with vRNA in the complex, resulting in the formation of large ER-derived structures (Fig. 11, Fig. 3). At this point, the distribution of vRNA would be determined by a balance between the formation and anchoring of the large structures and their spread towards the periphery of the cell. Our results are consistent with a model in which MP and microfilaments participate in the formation and anchoring of the ER-derived structures (Fig. 11, Fig. 3), while microtubules are involved in the transport to their final destinations; i.e., to the periphery for intercellular spread, or toward the nucleus for degradation (Fig. 11, Fig. 4).

Figure 11: Model of TMV infection in BY2 protoplasts. (1) Early in infection, TMV vRNA comes in close association with membranes of the ER, and vRNA-replicase complexes associated with ER are transported via microtubules to perinuclear positions. (2) Nascent vRNAs synthesize MP that remains associated with vRNA. (3) Formation and anchoring of large ER-derived structures containing MP, replicase, and vRNA that are stabilized by MP and microfilament interactions. (4) Microtubule-based transport system of vRNA-MP complexes towards the periphery of the cell to initiate cell-to-cell spread. (5) Protrusion of ER containing vRNA and MP through the plasma membrane. Note that the model is not drawn in scale.

Mentions:
The data presented here and in previous publications are consistent with a model of TMV infection (Fig. 11) in which the replication of vRNA takes place in close association with membranes of the ER (Fig. 11, Fig. 1). In this model, cytoskeletal elements are involved in targeting vRNA/replicase complexes to the perinuclear ER, perhaps via a retrograde flow of microtubules with ER attached as cargo. The ER-associated nascent vRNAs in replication complexes function as mRNAs for the synthesis of MP (Fig. 11, Fig. 2). The MP remains associated with vRNA in the complex, resulting in the formation of large ER-derived structures (Fig. 11, Fig. 3). At this point, the distribution of vRNA would be determined by a balance between the formation and anchoring of the large structures and their spread towards the periphery of the cell. Our results are consistent with a model in which MP and microfilaments participate in the formation and anchoring of the ER-derived structures (Fig. 11, Fig. 3), while microtubules are involved in the transport to their final destinations; i.e., to the periphery for intercellular spread, or toward the nucleus for degradation (Fig. 11, Fig. 4).

Bottom Line:
At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER.Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection.MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

ABSTRACTLittle is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.